Discovery of Anomer-Inverting Transglycosylase: Cyclic Glucohexadecaose-Producing Enzyme from Xanthomonas, a Phytopathogen

Various Xanthomonas species cause well-known plant diseases. Among various pathogenic factors, the role of α-1,6-cyclized β-1,2-glucohexadecaose (CβG16α) produced by Xanthomonas campestris pv. campestris was previously shown to be vital for infecting model organisms, Arabidopsis thaliana and Nicotiana benthamiana. However, enzymes responsible for biosynthesizing CβG16α are essentially unknown, which limits the generation of agrichemicals that inhibit CβG16α synthesis. In this study, we discovered that OpgD from X. campestris pv. campestris converts linear β-1,2-glucan to CβG16α. Structural and functional analyses revealed OpgD from X. campestris pv. campestris possesses an anomer-inverting transglycosylation mechanism, which is unprecedented among glycoside hydrolase family enzymes.

The positions of the mutations are underlined.

PDB entry 8X18
a Values in parentheses represent the highest resolution shell.

Note S5. Occupancy of 6-hydroxy group of the Glc moiety at subsite -16
The 6-OH group of the Glc moiety at subsite -16 seems to be able to move to the appropriate position for the reaction.The rotamer of the 6-OH in the co-crystal in this study is gt conformation, while the rotamer for the reaction is gg conformation.According to MD simulation by Abe et al 2 , both rotamers in 6-OH groups of Sopns exist comparably both in free and in complex structures.

Note S6. Differences in reaction mechanisms between GH enzymes and other enzymes (glycosyltransferases, glycoside phosphorylases and glycosynthases)
Structural, and functional analyses of XccOpgD revealed an anomer-inverting transglycosylation mechanism (Figure 1c).Among glycoside hydrolases, reactions are categorized into two canonical types, anomer-retaining GH and anomer-inverting GH (Figure 1).In both types, hydrolysis occurs when a nucleophile in the last step of the reaction is water (Figures 1a, b).In contrast, transglycosylation has been found only in an anomer-retaining mechanism 3 (Figure 1a).Therefore, the XccOpgD reaction (Figure 1c) is the first-discovered anomer-inverting transglycosylation.
Transglycosylation is a reaction catalyzing inter-or intra-molecular substitution of the anomeric position of glycosides 3 .Thus, the anomer-inverting transglycosylation should be clearly distinguished from the other anomer-inverting glycosyl transfer, namely forward reactions of glycosyltransferases 4- 6 , reverse reactions of glycoside phosphorylases 7 and artificially engineered glycosynthase 8 .These reactions depend on thermodynamical advantage (ability of leaving groups) of doner substrates such as sugar-mono or diphosphonucleotide, sugar 1-phosphate and sugar 1-fluoride.This is related to the fact that they have only one catalytic residue.In contrast, another catalytic residue is needed to make a nucleophile by attracting a proton from a hydroxy group of a glycoside (a poor leaving group) in an acceptor for transglycosylases.

NMR
The enzymatic reaction of 20 μg/mL XccOpgD, 1% linear β-1,2-glucan and 4 mM Tris-HCl buffer (pH 7.5) was incubated at 30°C overnight.Then, 10 μL 109 mg/mL BtBGL and 250 μL 1M bis-Tris buffer (pH 5.5) was added and incubated overnight.The reaction product was purified by sizeexclusion chromatography using a Toyopearl HW-40F column (~2 L gel).The sample was eluted with distilled water after the injection of the reaction mixture (~10 mL).The eluates were fractionated into 30-mL portions, and the fraction containing only the main product of the reaction by XccOpgD was lyophilized.One-( 1 H and 13 C) and two-dimensional (double-quantum-filtered COSY, heteronuclear single-quantum coherence (HSQC), heteronuclear multiple-bond correlation (HMBC) and HSQC-TOCSY) NMR spectra of the products were acquired in D2O at 298 K using a Bruker Avance 800 MHz spectrometer (Bruker, MA, USA) with tBuOH (δ 1.23 ppm for 1 H, and δ 31.30ppm for 13 C) as an internal standard.The H-1 chemical shift at the α-anomer was assigned by referring to the Karplus curve 14 .The H-2 chemical shift was assigned based on double-quantum-filtered COSY spectra.The C-1 and C-2 chemical shifts were assigned using HSQC spectra and based on the assignment of H-1 and H-2 proton signals.The H-6 and C-6 chemical shifts were determined using DEPT135, HSQC and HSQC-TOCSY data.Linkage positions between glucose units were determined by detecting cross-peaks in the HMBC spectrum.

General properties
The optimum pH of XccOpgD (0.1 mg/mL) activity was determined by incubating the protein in various 20 mM buffers (sodium acetate-HCl, pH 4.0-5.5;bis-Tris-HCl, pH 5.5-7.5;Tris-HCl, pH 7.5-9.0;glycine, pH 9.0-10.0)containing 0.25% linear β-1,2-glucan (average DP121) at 30 °C for 10 min and then heating at 100 °C for 5 min to terminate the reaction.It was confirmed that both Tris-HCl and bis-Tris-HCl did not affect the transglycosylation activity according to comparison with sodium phosphate.BtBGL and bis-Tris-HCl (pH 5.5) were added (final concentrations were 1 mg/mL and 83 mM, respectively) to convert non-cyclized β-1,2-glucan into glucose.The amounts of produced glucose were measured by the GOPOD method 15 to determine the amounts of residual linear β-1,2glucan.The conversion rates were calculated as subtracts between weights of the initial and residual substrates in the reaction by XccOpgD.The optimum temperature was determined by performing the reactions in 20 mM Tris-HCl buffer (pH 7.5) at each temperature (0-70 °C) for 10 min and then heated at 100 °C for 5 min to terminate the reaction.The conversion rates were calculated using the same approach to determine the optimum pH.

Kinetic analysis
The kinetic parameters for linear β-1,2-glucans were determined by performing the enzymatic reaction in a 20 μL reaction mixture containing 0.2 mg/mL XccOpgD, 0.007-0.), where v is the initial velocity, [E]0 is the enzyme concentration, [S] is the substrate concentration, kcat is the turnover number, and Km is the Michaelis constant.Each analysis was performed in triplicate, and the medians were used for regression.

Mutational analysis
The plasmids for producing XccOpgD mutants were constructed using a PrimeSTAR mutagenesis basal kit (Takara Bio) according to the manufacturer's instructions.PCRs were performed using appropriate primer pairs (Table S4) and the XccOpgD plasmid as the template.Transformation into E.
coli Rosseta2 (DE3) and the expression and purification of XccOpgD mutants were performed using the same methods described for wild-type XccOpgD preparation.The enzymatic reactions of XccOpgD mutants were performed similarly to determine the specific activity at 0.1 mM substrate concentration.The final assay concentration of the mutants and reaction times were 0.023-2.6mg/mL and 0-6.5 h, respectively, depending on the mutants.Colour development was performed in the same way as described in Kinetic analysis.

Figure S1 .
Figure S1.Electrospray ionization-mass spectrometry analysis.The peaks are assigned as [M + nNH4] n+ and indicated by arrows for the products.Green text represents forms of compounds (cyclic or linear) and degrees of polymerization (DPs) of products.For example, L5 represents a linear pentaose.The arrows next to the vertical axis represent the cutoffs which indicate the m/z value of the peak.a-e, ESI-MS data of linear β-1,2-glucan (a), the reaction products released from linear β-1,2glucan by XccOpgD (b), the reaction products after BtBGL treatment (c), purified CβG16α (d) and the reaction products released from CβG16α following treatment with BtBGL and CpSGL (e).

Figure S2 .
Figure S2.NMR analysis of the β-1,2-glucosidic bonds of the main product produced by XccOpgD.a, The 2D 1 H-1 H COSY spectrum of the main product from XccOpgD.Green and brown peaks represent COSY correlations between H1 and H2.b, Overlay of 2D HSQC-TOCSY and HMBC spectra of the main product from XccOpgD.Blue and purple peaks represent HSQC-TOCSY and HMBC correlations, respectively.The number labels beside the peaks are the numbers assigned for Glc moieties in Fig. 1c.Black lines trace from the 1C2 (2-carbon at the Glc moiety 1) to 2H1 (1-proton at the Glc moiety 2) in the direction to the non-reducing end.

Figure S4 .
Figure S4.Pairwise sequence alignment of XccOpgD and EcOpgD.The alignment was performed using Clustal Omega and visualized using the ESPript 3.0 server (http://espript.ibcp.fr/ESPript/ESPript/).The secondary structures of XccOpgD and EcOpgD are shown above and below the sequences.The regions of α-Helix 3 and Loop A are highlighted in blue letters.Blue and orange triangles represent substrate recognition residues via side chains and only main chains at subsites −7 to +6 of XccOpgD, respectively.

Figure S5 .
Figure S5.Superposition between Michaelis complexes of XccOpgD and EcOpgD.EcOpgD (PDB ID: 8IP1) is shown as a purple cartoon.Chains A and B of XccOpgD are shown as light green and cyan cartoons, respectively.Substrates are omitted.Chains A and B of EcOpgD form a closed state.In contrast, chains A and B of XccOpgD form closed and open states, respectively, because of crystal packing effects.The root mean square of deviation (RMSD) between XcOpgD and EcOpgD is 1.586 Å.

Figure S6 .
Figure S6.Superimposition between reaction centres of XccOpgD and EcOpgD.Chain A and the substrate of the XccOpgD complex structure (PDB ID: 8X18) are shown as light green and yellow sticks, respectively.Black and cyan labels represent residues in XccOpgD and EcOpgD, respectively.Chain A and water molecules of the superimposed EcOpgD complex structure (PDB ID: 8IP1) are shown as cyan lines and spheres, respectively.

Figure S8 .
Figure S8.Intramolecular interactions of the β-1,2-glucan at subsites −16 to −8.Substrates of subsites −16 to −8 and −7 to +6 are shown as yellow sticks and lines, respectively.Intramolecular hydrogen bonds are shown as dashed lines.Subsite positions are labelled with numbers.

Figure S9 .
Figure S9.Phylogenetic tree of GH186 and multiple sequence alignments.a, Each clade is indicated by a red circle with a clade number.Purple letters indicates the bootstrap values.Black arrows indicate clades, including homologues from Gram-negative bacteria whose phenotypes of OPG-related gene knockout mutants have been investigated.Asterisks represent species possessing two homologues.b-d, Multiple sequence alignments of clades 2-4.The alignments were performed using Clustal Omega (https://www.ebi.ac.uk/Tools/msa/clustalo/) and are visualized using the ESPript 3.0 server (http://espript.ibcp.fr/ESPript/ESPript/).The homologues are represented by UniProt accession numbers.Residue numbers of XccOpgD are shown above the alignments.
2 mM linear β-1,2-glucan and 20 mM Tris-HCl (pH 7.5) at 30 °C for 10 min.The reaction was stopped by heat treatment at 100 °C for 5 min.BtBGL and bis-Tris-HCl (pH 5.5) were added (final concentrations 1 mg/mL and 16.7 mM, respectively) to convert non-cyclized β-1,2-glucans into glucose.The reaction products were reduced using a one-fifth volume of 1 M NaBH4.The same volume of 1 M acetate as that of the 1M NaBH4 solution was added to each sample to neutralize NaBH4-treated solutions.The samples were then treated with 0.6 mg/mL of BtBGL and 0.15 mg/mL CpSGL at 30 °C for 24 h to convert CβG16α into 11 glucoses and a non-degradable glucopentaose.The main residual oligosaccharide was identified to be glucopentaose by ESI-MS.Colour development of the reaction mixtures was performed using the GOPOD method15 to quantify the concentration of glucose derived from the products released by XccOpgD.Molar concentrations of linear β-1,2-glucan substrates were calculated based on the Mn of the substrates.Kinetic parameters of XccOpgD were determined by fitting experimental data to the Michaelis-Menten equation, v/[E]0 = kcat [S]/(Km+ [S]

Table S3 . Specific activities of XccOpgD mutants
All specific activities were measured using 0.1 mM linear β-1,2-glucan as the substrate.Medians of triplicate experiments are presented.ND represents data of less than 0.00029 units/mg (0.1% relative activity).Maximum absolute values between medians and the other data of the triplicate experiments are shown in parentheses.Activity to produce 1 μmol of reaction product per minute is defined as 1 U (μmol/min).